The development of sensitive, precise, high throughput methods for directly monitoring specific proteins is increasingly desirable for diagnostic applications in both agricultural and clinical fields. Recent advances in the use of mass spectrometry (MS) in conjunction with protein/DNA-sequence database search-algorithms allow for the identification of proteins with unprecedented speed (Mann and Wilm 1994, Yates et al 1995). Despite these advances, it remains difficult to obtain accurate quantitative information concerning the levels of the identified proteins and the levels of site-specific or other modifications to individual protein molecules.
The principal state-of-the-art approaches for quantitative analysis of individual proteins are enzyme-linked immunosorbent assays (ELISAs) and sandwich ELISAs (sELISAs). The crux of these methods is the binding of an antibody molecule that recognizes the protein or peptide of interest. The development of an ELISA for a target protein is a particularly laborious and lengthy task, which requires production of monospecific antibodies to one or more epitopes residing within the protein. An epitope is a single limited amino acid sequence that is recognized by the antibody. ELISA systems based on a monoclonal antibody recognize a single epitope on the target protein ELISA systems based on polyclonal antibodies tend to recognize more than one epitope. The ELISA reaction requires binding of an antibody that may or may not be covalently linked to a group that generates enzymatic-derived colorimetric product or elicits fluorescence. These end-products of an ELISA are used for quantitation.
ELISA methods have several shortcomings. These assays are indirect in the sense that they require multiple steps to produce a product that is quantifiable. In addition, the occurrence of false positives and negatives is not uncommon. Thus, they may have insufficient sensitivity for commercial diagnoses, particularly in cases where there is significant risk of legal liability in the event of an incorrect result. Further, ELISA-based assays are limited in that they can only detect analytes for which antibodies have been raised. This requires prior knowledge of sample composition coupled with time-consuming effort in order to prepare sufficient purified protein and raise a new antibody for each target protein or peptide species. All of these factors generally prevent ELISA-based assays from being applied to identify previously unknown species or variant derivatives of a known species within a sample. Thus, ELISA systems are unable to detect subtle changes to a target protein that may have a dramatic effect on its physical and biological properties. For example, the antibody might not recognize a specific form of the protein or peptide that has been altered by post-translation modification such as phosphorylation or glycosylation, or conformationally obscured, or modified by partial degradation. Identification of such modifications is vital because changes in the physical and biological properties of these proteins may play an important role in their enzymatic, clinical or other biological activities. Such changes can limit the reliability and utility of ELISA-based quantification methods.
In the absence of appropriate antibodies, quantification is usually achieved by autoradiography after metabolic radiolabeling, fluorography, or the use of protein stains. These procedures depend on complete separation of the proteins of interest by techniques such as chromatographic separation or high-resolution two-dimensional electrophoresis (Boucherie et al. 1996).
In the late 1980's two new mass spectrometries became available for the analysis of large biomolecules, namely, matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS) and electrospray ionization (ESI). Requiring only a minute sample, mass spectrometry provides extremely detailed information about the molecules being analyzed, including high mass accuracy, and is easily automated. Both of these instruments are capable of mass analyzing biomolecules in complex biological solutions. MALDI-TOF MS involves laser pulses focused on a small sample plate comprising analyte molecules embedded in a low molecular weight, UV-absorbing matrix that enhances sample ionization. The matrix facilitates intact desorption and ionization of the sample. The laser pulses transfer energy to the matrix causing an ionization of the analyte molecules, producing a gaseous plume of intact, charged analyte. The ions generated by the laser pulses are accelerated to a fixed kinetic energy by a strong electric field and then pass through an electric field-free region in a vacuum in which the ions travel (drift) with a velocity corresponding to their respective mass-to-charge ratios (m/z). The lighter ions travel through the vacuum region faster than the heavier ions thereby causing a separation. At the end of the electric field-free region, the ions collide with a detector that generates a signal as each set of ions of a particular mass-to-charge ratio strikes the detector. Travel time is proportional to the square root of the mass as defined by the following equation t=(m/(2KE)z)1/2 where t=travel time, s=travel distance, m=mass, KE=kinetic energy, and z=number of charges on an ion. A calibration procedure using a reference standard of known mass can be used to establish an accurate relationship between flight time and the mass-to-charge ratio of the ion. Ions generated by MALDI exhibit a broad energy spread after acceleration in a stationary electric field. Forming ions in a field-free region, and then applying a high voltage pulse after a predetermined time delay (e.g. “delayed extraction™”) to accelerate the ions can minimize this energy spread, which improves resolution and mass accuracy.
In a given assay, 50 to 100 mass spectra resulting from individual laser pulses are summed together to make a single composite mass spectrum with an improved signal-to-noise ratio. The entire process is completed in a matter of microseconds. In an automated apparatus, tens to hundreds of samples can be analyzed per minute. In addition to speed, MALDI-TOF technology has many advantages, which include: 1) mass range—where the mass range is limited by ionization ability, 2) complete mass spectrum can be obtained from a single ionization event (also referred to as multiplexing or parallel detection), 3) compatibility with buffers normally used in biological assays, 4) very high sensitivity; and 5) requires only femtomoles of sample. Thus, the performance of a mass spectrometer is measured by its sensitivity, mass resolution, and mass accuracy.
In order for mass spectrometry to be a useful tool for detecting and quantifying proteins, several basic requirements need to be met. First, targeted proteins to be detected and quantified must be concentrated (e.g., enriched and/or fractionated) in order to minimize the effects of salt ions and other molecular contaminants that reduce the intensity and quality of the mass spectrometric signal to a point where either the signal is undetectable or unreliable, or the mass accuracy and/or resolution is below the value necessary to detect the target protein. Second, mass accuracy and resolution significantly degrade as the mass of the analyte increases. Thus, the size of the target protein or peptide must be within the range of the mass spectrometry device where there is the necessary mass resolution and accuracy. Third, to be able to quantify accurately, one would preferably resolve the masses of the peptides by at least six Daltons to increase quality assurance and to prevent ambiguities. Fourth, the mass spectrometric methods for protein detection and quantification diagnostic screening must be efficient and cost effective in order to screen a large number of samples in as few steps as possible.
Mass spectrometry methods for the quantitation of proteins in complex mixtures have employed a system using protein reactive reagents comprised of three moieties that are linked covalently; an amino acid reactive group, an affinity group and an isotopically tagged linker group (Aebersold et al, 2000). This class of new chemical reagents is referred to as Isotope-Coded Affinity Tags (ICATs) (Gygi et al 1999). The reactive group embodied used sulfhydryl groups that react specifically with the amino acid cysteine. The presence of the affinity group facilitates the isolation of the specifically labeled proteins or peptides from a complex protein mixture. Selected affinity groups include strepavidin or avidin. Only those proteins containing these affinity groups may be isolated. The linker moiety may be isotopically labeled by a variety of isotopes that include 3H, 13C, 15N, 17O, 18O and 34S. The use of differential isotopic ICATs provides a method for the quantitation of the relative concentration of peptides in different samples by mass spectrometry. The methods can be used to generate global protein expression profiles in cells and tissues exposed to a variety of conditions.
In an analogous method, the N-terminal amino acids of proteins from two states are differentially labeled using different isotopically tagged nicotinyl-N-hydroxysuccinimide reagents (Munchbach et al, 2000). Unlike the ICAT system, proteins are first separated by two-dimensional SDS polyacrylamide gel electrophoresis before the analysis is performed. The ratio of the isotope for each protein determined by mass spectrometry provides a relative concentration of each protein present in different physiological states.
It is believed that the limitations of mass spectrometry methods employing either ICATs or N-succinylation isotopic tagging are inherently associated with the requirement that the protein from one sample is quantified relative to another state or sample rather than being quantified in absolute amounts. In the case of the ICAT method, it is a requirement that the protein or peptide being quantified contains at least one amino acid that is modified by the reactive group. A related requirement is that the reactive amino acid site on the protein in the two or more states or samples must be equivalently accessible to the reactive group on the ICATs. Similar to antibody methods, if the site is altered or conformationally obscured then the quantitation of the protein will be compromised. An additional limitation in the use of N-succinylation of proteins is that it requires the laborious task of two-dimensional SDS polyacrylamide gel electrophoresis prior to analysis.
There remains a pressing need for easier, more reliable means to rapidly detect, quantify and characterize specific proteins from biologically complex samples.